Distribution and structural diversity of Type IV internal ribosome entry sites

Internal ribosome entry sites (IRESs) are RNA sequences that facilitate cap- and end-independent translation initiation in eukaryotes. Type IV IRESs, which include the hepatitis C virus IRES, directly bind the 40S ribosomal subunit and require only a subset of canonical initiation factors to function. As the full extent of diversity and species distribution of type IV IRESs was unknown, we sought to identify and classify the architectural variation of all members. Using a secondary structure homology-based search method, we identified 163 putative type IV IRESs from viruses with diverse hosts and phylogeny, including the first example in a double-stranded viral genome. Clustering analysis based on the presence and overall size of secondary structure elements yielded three distinct groups, differentiated by substantial expansions and deletions. Chemical probing of representative IRES RNAs from each cluster confirmed predicted secondary structures. Subsequent in vitro translation assays suggested that structural differences produce functional variation. Our findings reveal distinct structural adaptations and patterns within the type IV IRESs that may influence IRES function and mechanism.